Queuine (one of ca. 100 naturally occurring modified bases) is found in the wobble position (34) of 4 tRNAs (anticodons GUN) in eubacteria and eukaryotes. It is introduced into tRNAs by a post-transcriptional base exchange catalyzed by tRNA-guanine transglycosylase (TGT, also conserved across the kingdoms).. It has been shown that TGT is required for pathogenicity of the dysentery-causing bacterium Shigella flexneri. While the tgt gene has been shown not to be required for growth in cell culture, the relationship between tgt and pathogenicity is not understood, and the present data suggest that TGT could be a novel antibiotic target. Previous studies have elucidated many aspects of the molecular basis of the recognition and discrimination of specific tRNAs and heterocyclic substrates by the eubacterial TGT. However, much work still needs to be done to understand the differential properties of eukaryotic vs. eubacterial TGTs. Other than correlations with proliferation, differentiation, retroviral frameshifting, and bacterial virulence, little is known about the physiological roles of queuine let alone how these roles are carried out. There are two overarching goals of this proposal. The first is to elucidate the similarities and differences between the eubacterial TGT and the human TGT. These studies are prerequisite to the rational design of selective inhibitors of the eubacterial TGT. Our second overarching goal is to try to elucidate the physiological role(s) of queuine in eubacteria and eukaryotes. Given the potential of TGT as a novel antibiotic target, it is important to understand the physiological role(s) of queuine and any differences between human and eubacteria. In order to achieve these goals, we have proposed a number of specific aims that bring many techniques and approaches (e.g., molecular biology, enzymology, crystallography, computational chemistry, synthetic chemistry, genomics, and proteomics) to bear on this problem. To carry out these studies in a rigorous fashion, we have established collaborations with key experts and take advantage of the resources available here at the University of Michigan. A clear understanding of the similarities and differences between the TGTs from eubacteria and human will be of significance for many reasons but most obviously for the future design of novel antibiotics.